Type 1 allergies are of importance worldwide. Up to 20% of the population in industrialised countries suffer from complaints such as allergic rhinitis, conjunctivitis or bronchial asthma. These allergies are caused by allergens present in the air (aeroallergens) which are liberated from sources of various origin, such as plant pollen, mites, cats or dogs. Up to 40% of these type 1 allergy sufferers in turn exhibit specific IgE reactivity with grass pollen allergens (Freidhoff et al., 1986, J. Allergy Clin. Immunol. 78, 1190-2002).
The substances which trigger type 1 allergy are proteins, glycoproteins or polypeptides. After uptake via the mucous membranes, these allergens react with the IgE molecules bonded to the surface of mast cells in sensitised individuals. If two IgE molecules are crosslinked to one another by an allergen, this results in the release of mediators (for example histamine, prostaglandins) and cytokines by the effector cell and thus in the corresponding clinical symptoms.
A distinction is made between major and minor allergens depending on the relative frequency with which the individual allergen molecules react with the IgE antibodies of allergy sufferers.
In the case of timothy grass (Phleum pretense), Phl p 1 (Petersen et al., 1993, J. Allergy Clin. Immunol. 92: 789-796), Phl p 5 (Matthiesen and Löwenstein, 1991, Clin. Exp. Allergy 21: 297-307; Petersen et al., 1992, Int. Arch. Allergy Immunol. 98: 105-109), Phl p 6 (Petersen et al., 1995, Int. Arch. Allergy Immunol. 108, 49-54). Phl p 2/3 (Dolecek et al., 1993, FEBS 335 (3), 299-304), Phl p 4 (Haavik et al., 1985, Int. Arch. Allergy Appl. Immunol. 78: 260-268; Valenta et al., 1992, Int. Arch. Allergy Immunol. 97: 287-294, Fischer et al., 1996, J. Allergy Clin. Immunol. 98: 189-198) and Phl p 13 (Suck et al., 2000, Clin. Exp. Allergy 30: 324-332; Suck et al., 2000, Clin. Exp. Allergy 30: 1395-1402) have hitherto been identified as major allergens.
The dominant major allergens of timothy grass (Phleum pretense) are Phl p 1 and Phl p 5, with Phl p 5 occurring in two forms 5a and 5b which differ in respect of their molecular weight and are encoded by independent genes. The deduced amino acid sequences both of Phl p 5a and also of Phl p 5b have been determined by means of the recombinant DNA technique. Phl p 5a is a protein of about 32 kDa and reacts with the IgE antibodies of 85-90% of grass pollen allergy sufferers. Phl p 5a exists in a series of homologous variants which differ from one another through point mutations and probably correspond to different allelic forms. The pollen of related grass species, such as, for example, Lolium perenne, Poa pratensis inter alia, contains allergens which are homologous with that of Phl p 5a and together are known as group 5 allergens. The high structural homology of these group 5 allergens of grass species causes correspondingly high cross reactivity of the molecules with the IgE antibodies of grass pollen allergy sufferers.
A classical approach to effective therapeutic treatment of allergies is specific immunotherapy or hyposensitisation (Fiebig, 1995, Allergo J. 4 (6): 336-339, Bousquet et al., 1998, J. Allergy Clin. Immunol. 102 (4): 558-562). In this method, the patient is injected subcutaneously with natural allergen extracts in increasing doses. However, there is a risk in this method of allergic reactions or even anaphylactic shock. In order to minimise these risks, innovative preparations in the form of allergoids are employed. These are chemically modified allergen extracts which have significantly reduced IgE reactivity, but identical T-cell reactivity compared with the untreated extract (Fiebig, 1995, Allergo J. 4 (7): 377-382).
Even more substantial therapy optimisation would be possible with allergens prepared by recombinant methods. Defined cocktails of high-purity allergens prepared by recombinant methods, optionally matched to the individual sensitisation patterns of the patients, could replace extracts from natural allergen sources since these, in addition to the various allergens, contain a relatively large number of immunogenic, but non-allergenic secondary proteins.
Realistic perspectives which may result in reliable hyposensitisation with recombinant expression products are offered by specifically mutated recombinant allergens in which IgE epitopes are specifically deleted without impairing the T-cell epitopes which are essential for therapy (Schramm et al., 1999, J. Immunol. 162: 2406-2414).
A further possibility for therapeutic influencing of the disturbed T helper cell equilibrium in allergy sufferers is treatment with expressible DNA which encodes for the relevant allergens (immunotherapeutic DNA vaccination). Initial experimental evidence of allergen-specific influencing of the immune response by a DNA vaccine of this type has been furnished in rodents by injection of allergen-encoding DNA (Hsu et al., 1996, Nature Medicine 2 (5): 540-544).
The object on which the present invention is based consisted in the provision of novel variants of the group 5 allergens of the Pooideae at the protein and DNA level which are distinguished by reduced IgE activity at the same time as substantial retention of the T-cell reactivity and are therefore suitable for specific immunotherapy and immunotherapeutic DNA vaccination.